The subject matter of the present invention is a radiopharmaceutical product using radioactive metal complexes for the selective labeling of lymphocytes.
Such radioactive products are suitable in particular for the diagnosis of inflammatory processes and of malignant lymphomas, and for radiotherapy of malignant lymphomas.
Radiolabeled products used for these diagnoses and therapies are described in particular in the work: Radiolabeled Blood Elements, published by J. Martin-Conin, Plenum Press, New-York, 1994, pages 261 to 263 [1] and pages 265 to 271 [2].
In these documents, lymphocytes labeled with 111In-oxinetropolone, lymphocytes labeled with 99mTc-HMPAO, nanocolloids labeled with 99mTc-HSA, polyclonal human immunoglobulins or monoclonal antibodies are used.
These techniques do not give satisfactory results since it is necessary firstly to separate the lymphocytes from whole blood in order to label them with appropriate products.
More recently, it has been found that radiopharmaceutical products containing bis(dithiocarboxylato)-nitrurotechnetium-99m could be used to achieve selective labeling of leukocytes, and studies have been made on the influence of the hydrocarbon chain of the ligand on the results obtained, as described in Nuclear Medicine and Biology, 1997, volume 24, pages 439-445 [3] and 1999, volume 26, pages 225-231, [4]. Radiopharmaceutical products of this type are of great interest since when they placed in the presence of blood cells (erythrocytes, leukocytes, platelets) these radioactive complexes show selective affinity for leukocytes. Also, document [4] showed that the complex [99mTCN(CH3)(CH2)8CS2)2] also has selectivity for lymphocytes.
Contrary to radiolabeled granulocytes, which are routinely used in clinical practice to detect sites of infection and/or inflammation, radiolabeled lymphocytes have been little used either for diagnostic or for therapeutic purposes.
This is due to the combination of several technical or basic difficulties. Among operative difficulties, the need must be taken into account to make prior separation, before radiolabeling, of the lymphocytes from the other blood cells in whole blood, in particular from the granulocytes.
Despite the difficulties mentioned, radiolabeled lymphocytes could be used in man for improved diagnosis and for improved therapeutic management of diseases characterized by chronic inflammation.
Also, recently published results have shown that malignant lymphomas, in particular non-Hodgkin""s lymphoma, could be successfully treated by radioimmunotherapy, a technique which consists of using a monoclonal antibody radiolabeled with xcex2xe2x88x92 ray emitters having a strong affinity for malignant lymphocytes. In this way the lymphocytes are radiated by the xcex2xe2x88x92 ray emission of the radioelement vectored by the antibody which binds itself to the surface of the cells. Radioimmunotherapy of lymphomas, however, encounters various difficulties, including the use of large quantities of non-labeled antibodies to saturate the high number of antigen sites present on the cells. The cost of treatment is therefore greatly increased. In addition, there is a risk that the injection of the first therapeutic dose may cause an immunity reaction which creates human antibodies directed against the antibodies used for treatment (anti-antibodies). If a second therapeutic dose is required in the same patient, it will be much less effective since the labeled antibody would be inhibited by the presence of anti-antibody antibodies. The use of a compound having a simple chemical structure which binds selectively to lymphocytes would avoid these types of problems.
The subject matter of the present invention is precisely radiopharmaceutical products using complexes having a simple chemical structure which do not give rise to the above-mentioned disadvantages.
The subject matter of the present invention is a radiopharmaceutical product characterized in that it contains a radioactive metal complex meeting the formula:
[M(R1CS2)(R1CS3)2]
in which M is chosen from among 99mTc, 186Re and 188Re, and R1 represents an alkyl, cycloalkyl, aralkyl or aryl group, which may or may not be substituted by one or more substituents chosen from among the halogen atoms, the hydroxyl group, the alkyl groups and the alcoxy groups.
In this radiopharmaceutical product, the radioactive metal M which may be 99mTc for diagnosis, or 186Re or 188Re for therapy, is coordinated by an assembly of non-homogeneous sulfured ligands, namely a dithiocarboxylate ligand (R1CS2)xe2x88x92 and two trithioperoxycarboxylate ligands (R1CS3)xe2x88x92. In this structure, the radioactive metal is in M3+ form.
The document: Inorganic Chemistry, 1997, vol. 36, pages 6144-6145 [5] describes a rhenium complex having the same type of structure but which is not obtained from 186Re or 188Re. It is therefore not a radiopharmaceutical product.
In the structure of the complexes of the invention, the R1 groups of the sulfured ligands may be alkyl, cycloalkyl, aralkyl or aryl aliphatic groups. These groups may be non-substituted or substituted by one or more substituents chosen from among the halogen atoms, fluorine for example, the hydroxyl group, the alkyl groups and the alcoxy groups.
The alkyl groups used for R1 may be linear or branched groups at C1 to C15, preferably groups having 3 to 13 carbon atoms.
The cycloalkyl groups used for R1 preferably have 3 to 7 carbon atoms, for example 6 carbon atoms.
The aryl groups used for R1 may be of phenyl or naphthyl type.
The aralkyl groups used for R1 may be of C6H5(CH2)n type in which n ranges from 1 to 3, preferably n equals 1 or 2.
Preferably, in accordance with the invention, the R1 group is an aryl, aralkyl or cyclohexyl group that is optionally substituted.
Advantageously, when R1 is an aryl group, it is chosen from among the phenyl groups, phenyl substituted by a methyl, ethyl, butyl, ethoxy, methoxy or hydroxyl group, phenyl substituted by a fluorine atom, phenyl substituted by three methyl groups, naphthyl and naphthyl substituted by a methyl group.
When R1 is an aralkyl group, this is advantageously the benzyl or phenethyl group.
A further purpose of the invention is a method for preparing a radiopharmaceutical product containing a radioactive metal complex having the formula:
[M(R1CS2)(R1CS3)2]
in which M is chosen from among 99mTc, 186Re and 188Re, and R1 represents an alkyl, cycloalkyl, aralkyl or aryl group, which may or may not be substituted by one or more substituents chosen from among the halogen atoms, the hydroxyl group, the alkyl groups and the alcoxy groups,
which consists of causing a salt of formula (MO4)xe2x88x92Z1+, in which M is as defined above and Z1 is a pharmaceutically acceptable cation, to react with a reducing agent, and of adding to the reaction mixture a dithiocarboxylate of formula (R1CS2)xe2x88x92Z2+ in which R1 is as defined above and Z2+ represents a pharmaceutically acceptable cation.
The pharmaceutically acceptable cations used for Z1 may be ions of alkali or alkaline earth metals, for example Na.
The pharmaceutically acceptable cations used for Z2 may be chosen from among MgX+ in which X is a halogen atom such as Br or Cl, the quaternary ammonium cations, and the ions of alkaline earth metals such as Na.
The quaternary ammonium cations may for example be of NR4 type, in which R is an alkyl group, methyl for example. It is also possible to use quaternary ammonium cations of piperidinium type having the formula C5H10ONH2+.
In the method of the invention, the reducing agent used may be of various types. In particular a reducing agent may be used which is made up of a tin salt associated with a complexing agent having complexing properties for tin that are greater than those of dithiocarboxylate. This complexing agent may be of phosphonate, polyphosphate and polyaminocarboxylic acid type. By way of example of such complexing agents, mention may be made of pyrophosphates of ammonium or of an alkali or alkaline earth metal, glucoheptonates of ammonium or of an alkali or alkaline earth metal, diethylene triaminopentacetates of ammonium or of an alkali metal, ethylene diaminotetracetates of ammonium or of an alkali or alkaline earth metal, 1,2-diaminopropane-N,N,Nxe2x80x2,Nxe2x80x2-tetracetates of ammonium or of an alkali or alkaline earth metal, gluconates of ammonium or of an alkali or alkaline earth metal, methylene diphosphonates of ammonium or of an alkali or alkaline earth metal, hydroxymethylene diphosphonates of ammonium or of an alkali or alkaline earth metal, citrates of ammonium or of an alkali or alkaline earth metal.
By way of example, it is possible in the method of the invention to use a tin salt made up of tin chloride associated with a complexing agent chosen from among calcium gluconate and 1,2-diaminopropane-N,N,Nxe2x80x2Nxe2x80x2-tetracetic acid.
According to the invention it is also possible to used reducing agents made up of triphenylphosphine or one of its derivatives associated with hydrochloric acid.
As an example of derivative of triphenylphosphine, mention may be made of sodium triphenylphosphine-tri-meta-sulfonate P(C6H4SO3)3Na3.
In the method of the invention, the metal M initially at oxidation state 7 is reduced to oxidation state 3, while part of the dithiocarboxylate ligand is oxidized to trithioperoxycarboxylate.
The quantities of reducing agent used with this method are chosen in relation to the quantity of pertechnetate or perrhenate initially added.
In respect of 99mTc pertechnetate , for activities ranging from 30 MBq to 4 GBq, the quantities of reducing agent used may range from 0.01 to 1 mg for SnCl2, 2H2O, in the presence of an excess of complexing agent relative to the tin chloride.
When a triphenylphosphine is used as reducing agent, the quantities used are in the order of 0.1 to 5 mg for pure triphenylphosphine, and 0.2 to 10 mg for sodium triphenylphosphine trisulfonate. With these reducing agents, an aqueous solution of HCl is added to obtain 1.10xe2x88x922 to 1.10xe2x88x921 mol/L of HCl in the reaction medium.
Despite the similarity in the chemical properties of pertechnetate and perrhenate, it is known that for the reduction reaction, the latter ion requires greater quantities of reducing agent than those used for the pertechnetate ion.
In addition, when the radioactive metal is 186rhenium, an isotope having low specific activity, the quantity of perrhenate used is greater to obtain the same activity; therefore, to reduce this species, greater quantities of reducing agent are used than for the isotope 188rhenium.
Therefore, 0.1 to 5 mg of reducing agent may be used for SnCl2, 2H2O, from 0.1 to 10 mg for pure triphenylphosphine, and from 0.2 to 20 mg for sodium triphenylphosphine trisulfonate.
A sufficient quantity of dithiocarboxylate, preferably dissolved in physiological serum, is then added to the reaction medium. The reaction of the ligand with the pertechnetate or perrhenate is conducted under heat, for example at a temperature of 100xc2x0 C.
A further purpose of the invention is a kit for the preparation of a radiopharmaceutical product containing a radioactive metal complex having the formula:
[M(R1CS2)(R1CS3)2]
in which M is chosen from among 99mTc, 186Re and 188Re, and R1 represents an alkyl, cycloalkyl, aralkyl or aryl group, which may or may not be substituted by one or more substituents chosen from among the halogen atoms, the hydroxyl group, the alkyl groups and the alcoxy groups,
characterized in that it comprises:
a first bottle containing
a) a tin salt associated with a complexing agent, or
b) a triphenylphosphine and hydrochloric acid, and
a second bottle containing dithiocarboxylate with the formula (R1CS2)xe2x88x92Z2+ in which R1 is as defined above and Z2 is a pharmaceutically acceptable cation.
According to one first embodiment of the kit, the first bottle comprises tin chloride SnCl2, 2H2O associated with a complexing agent chosen from among calcium gluconate and 1,2-diaminopropane-N,N,Nxe2x80x2,Nxe2x80x2-tetracetic acid.
According to a second embodiment of the kit, the first bottle contains triphenylphosphine or sodium triphenylphosphine-trisulfonate, and hydrochloric acid.
The radiopharmaceutical products described above which selectively attach themselves to lymphocytes, may be used in compositions for the diagnosis of inflammatory processes when M is 99mTc, or in compositions for the radiotherapy of malignant lymphomas if M is 186Re or 6188Re.